In order to provide prophylactic protection against HIV, a successful vaccine will need to elicit mucosal immunity, in order to fight the virus at the mucosal sites that are major points of entry. Among the potential routes for vaccines to promote mucosal immunity, oral delivery is of great interest as a simple, needle-free mode of administration, but is made challenging by the need to protect vaccines during transit through the gastrointestinal tract and achieve sufficient uptake of the vaccine by the mucosal tissue. We have recently developed a polymeric vaccine system comprised of chemokines and antigen-loaded nanoparticles co-delivered within alginate hydrogel microsphere carriers. Alginate microspheres are candidate materials for oral drug delivery due to their stability at low pH, and alginate particles have previously been reported to promote mucosal immunity via oral vaccination. Thus, we propose that the alginate microsphere carriers we have developed can (1) protect antigen/co-delivered chemokine (or cytokine) during transit through the gastrointestinal (Gl) tract and (2) promote uptake by the gut-associated lymphoid tissues (GALT). Microparticles are known to be transcytosed by M cells overlying Peyer's patches, and extracellular deposition of alginate carriers in the GALT would allow for local generation of dendritic cell (DC)-attracting chemokine gradients that would draw DCs to these microdepots of antigen-loaded nanoparticles. We propose here to perform initial tests of this concept for the generation of oral vaccines that could prime mucosal immunity to HIV. The specific aims are: Aim 1: We will characterize the ability of alginate microsphere carriers prepared by different means to protect antigen delivery nanoparticles and co-delivered chemokines from simulated gastric fluid in vitro and to be taken up by gut mucosa in vivo, and characterize GALT uptake of alginate carriers as a function of particle size. Aim 2: Using optimal carrier sizes and compositions defined from the studies in Aim 1, we will measure immune responses triggered by nanoparticles delivering gp120 antigen and CpG alone or with co-delivery of the chemokine CCL20, and analyze antigen dissemination and dendritic cell trafficking in response to these vaccine particles. The studies described will enable the rationale design of microsphere carriers that can promote strong mucosal and systemic immune responses to recombinant HIV antigens, as a step toward a prophylactic HIV vaccine. [unreadable] [unreadable] [unreadable]